In Vitro Microevolution and Co-Selection Assessment of Amoxicillin and Cefotaxime Impact on Escherichia coli Resistance Development

Abstract

The global spread of antimicrobial resistance has become a prominent issue in both veterinary and public health in the 21st century. The extensive use of amoxicillin, a beta-lactam antibiotic, and consequent resistance development are particularly alarming in food-producing animals, with a focus on the swine and poultry sectors. Another beta-lactam, cefotaxime, is widely utilized in human medicine, where the escalating resistance to third- and fourth-generation cephalosporins is a major concern. The aim of this study was to simulate the development of phenotypic and genotypic resistance to beta-lactam antibiotics, focusing on amoxicillin and cefotaxime. The investigation of the minimal inhibitory concentrations (MIC) of antibiotics was performed at 1×, 10×, 100×, and 1000× concentrations using the modified microbial evolution and growth arena (MEGA-plate) method. Our results indicate that amoxicillin significantly increased the MIC values of several tested antibiotics, except for oxytetracycline and florfenicol. In the case of cefotaxime, this increase was observed in all classes. A total of 44 antimicrobial resistance genes were identified in all samples. Chromosomal point mutations, particularly concerning cefotaxime, revealed numerous complex mutations, deletions, insertions, and single nucleotide polymorphisms (SNPs) that were not experienced in the case of amoxicillin. The findings suggest that, regarding amoxicillin, the point mutation of the acrB gene could explain the observed MIC value increases due to the heightened activity of the acrAB-tolC efflux pump system. However, under the influence of cefotaxime, more intricate processes occurred, including complex amino acid substitutions in the ampC gene promoter region, increased enzyme production induced by amino acid substitutions and SNPs, as well as mutations in the acrR and robA repressor genes that heightened the activity of the acrAB-tolC efflux pump system. These changes may contribute to the significant MIC increases observed for all tested antibiotics. The results underscore the importance of understanding cross-resistance development between individual drugs when choosing clinical alternative drugs. The point mutations in the mdtB and emrR genes may also contribute to the increased activity of the mdtABC-tolC and emrAB-tolC pump systems against all tested antibiotics. The exceptionally high mutation rate induced by cephalosporins justifies further investigations to clarify the exact mechanism behind.

Monitoring Changes in the Antimicrobial-Resistance Gene Set (ARG) of Raw Milk and Dairy Products in a Cattle Farm, from Production to Consumption

“…There are natural limiting factors in the transfer of ARGs [ 71 ], among which the most important is the shared physical space, known as the minimal critical density of bacterial populations [ 72 ]. Another limiting factor is the energy cost associated with carrying individual ARGs and the mechanisms they express, and the maintenance of these genes will only be successful if it confers an evolutionary advantage to the bacteria [ 71 , 73 , 74 , 75 ]. Acquisition of resistance genes from the gut microbiome via milk or dairy products can only occur through transformation and transduction if they are pasteurized; however, the high bacterial load in raw milk and dairy products increases the likelihood of conjugation, which poses a much greater risk in the spread of antimicrobial resistance [ 76 ].…”

Stability Studies of the Dilution Series of Different Antibiotic Stock Solutions in Culture Medium Incubated at 37 °C

“…The gold-standard methods for such assessments—the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)—vary in duration, lasting anywhere from 24 h to several days, depending on the organism involved. These evaluations are conducted across different culture media [ 4 , 5 ], with evolution and co-selection studies extending up to 10–12 days [ 6 , 7 , 8 ]. Therefore, particularly for extended-duration studies, an understanding of the stability of various antibiotics in both ultrapure water (UPW) and culture media is essential, as it can significantly influence experimental outcomes.…”